MBL is a serum protein involved in innate immunity. The molecular weight is 32 KD, consisting of carboxy-terminal carbohydrate binding domain (CRD), collagen domain, and amino-terminal cysteine-rich region. The collagen domains of three MBL molecules form a triple helix resulting in a formation of a trimer, and then up to six units of the trimers form a giant molecule by inter molecular disulfide bonding using amino-terminal cysteines.
The MBL is associated with other proteins such as MBL associated serine proteases (MASP 1, MASP 2, or MASP 3) or MBL associated protein (Map19). Therefore, the overall molecular shape of MBL is similar to the first component of complement system (C1q). The function of activated MBL is also similar to C1q, but unlike C1q it activates complement system by cleaving C4 and C2. Activation of MBL requires binding of microorganisms with unique glycosylation pattern on their surface proteins. In this process the MBL associated serine protease is activated, and C4 and C2 are cleaved in a similar way that C1q associated serine proteases (C1r and C1s) triggers activation of complement system by cleaving C3. MBL binding to microorganism also prepares the microorganism for efficient phagocytosis, as if it is an opsonins.
Previously MBL gene has been expressed in various cell lines, including CHO cell (Katsuki Ohtani et al, J. of Inmunol. Methods 222, 135–144, 1999) or HLF hepatoma cell line or HEK 293 EBNA cell (T. Vorup-Jensen et al, International Immunopharmacology 1, 677–687, 2001).
High yield expression was possible in a CHO cell, but the MBL recovered was mainly monomers and dimers without significant amount of oligomers. Expression of MBL gene in transformed human cell lines produced significantly more oligomers, but the over all production yield was less than 1 ug/mL culture media.
It is well known that only high molecular weight form of MBL complex is capable of activating complement system. Activation of complement system is very important in defense against microbial infection. It not only prepares for invading microorganism for efficient phagocytosis and direct lysis, but also it helps efficient induction of adaptive immunity.
Serum level of MBL varies widely in different individuals, ranging from 50 ng/mL or lower to over 3 ug/mL serum mainly due to genetic variation. Genetic variation includes point mutations on exons and mutations on promoter regions. Generally individuals with low level of MBL are susceptible from microbial infection. Specially, those newborn children with low level of MBL are dangerously susceptible from infections. According to one survey (Y. Hakozaki et al, Liver, 22, 29–34, 2002), the mortality of patients going through hepatic failure due to Hepatitis B virus infection depended on the serum level of MBL. Patients with high level of MBL (3 ug/mL) did not die, whereas 80% of the patients with low MBL level died. Therefore those individuals with low MBL level might benefit from MBL supplement.
Therapeutic use of recombinant MBL requires not only high level expression of MBL for industrial quantity production but also high molecular oligomeric form of MBL.